The present invention relates in general to the field of medical diagnostics, and more particularly to assays for determining cartilage degeneration status, including cartilage degeneration status in osteoarthritis (“OA”), rheumatoid arthritis (“RA”) status, and status in other arthritic conditions.
The hallmark of OA, the most common cartilage degeneration joint disease, and RA is cartilage loss leading to joint destruction. Knee OA, one of the most common forms of OA, is associated with significant morbidity (Felson, D. T., Epidemiology of Osteoarthritis. In: Brandt K. F., et al., eds., OSTEOARTHRITIS. Oxford University Press, pp. 13-22 (1998)).
To assess the progression of cartilage destruction the most established methods are the measurement of joint space width (JSW) using plain X-rays and the assessment of chondropathy by arthroscopic evaluation of the knee. These two techniques have however some limitations. When there is radiological evidence of OA, there is often already significant joint damage. Because changes of JSW are relatively small compared to the precision error of X-ray measurements, at least one year and preferably 2 years are usually necessary to accurately assess the progression of joint damage or its reduction by treatment (Ravaud, P., et al., Variability in knee radiographing: implication for definition of radiological progression in medial knee osteoarthritis, Ann. Rheum. Dis. 57:624-629 (1998)). Magnetic resonance imaging is more sensitive than plain X-ray, although its reproducibility is not yet fully validated and is currently being optimized for monitoring patients with OA.
Arthroscopy provides a direct and magnified view of the cartilage surface that has prompted some to consider arthroscopy as the gold standard for the assessment of cartilage lesions (Fife, R. S., et al., Relationships between arthroscopic evidence of cartilage damage and radiographic evidence of joint space narrowing in early osteoarthritis of the knee, Arthritis Rheum. 34:377-382 (1991); Ayral, X., et al., Chondroscopy: a new method for scoring chondropathy, Semin. Arthritis Rheum. 22:289-297 (1993)). Arthroscopic scoring systems of chondropathy have been established and validated (Aryal, X., Semin. Arthritis Rheum. 22, supra; Ayral, X., et al., Arthroscopic evaluation of chondropathy in osteoarthritis of the knee, J. Rheumatol. 23:698-706 (1996)). This is however an invasive technique which can not be routinely applied to all patients and which requires trained investigators. Clearly, for identifying patients at high risk for destructive OA and for monitoring drug efficacy there is a need for non-invasive methods that can be repeated and have improved sensitivity compared to plain X-rays.
Molecular markers are molecules or fragments thereof of tissue matrices which are released into biological fluids during the process of tissue biosynthesis and turnover and which can be measured by immunoassays. Molecular markers of bone, cartilage and synovium have been described and their changes have been investigated in patients with OA, mainly in cross-sectional studies (Garnero, P., et al., Molecular basis and clinical use of biochemical markers of bone, cartilage and synovium in joint diseases, Arthritis Rheum. 43:953-961 (2000)). However, detection of molecular markers for collagen synthesis or degradation has not been used to provide information on the progression of OA and other forms of cartilage degeneration.
Because the loss of cartilage is believed to result from the combination of a decreased reparative process coupled with an increased degradative phenomenon (Dean, D. D., et al., Evidence for metalloproteinase and metalloproteinase inhibitor imbalance in human osteoarthritic cartilage, J. Clin. Invest. 84:678-685 (1989); Poole, A. R., Cartilage in health and disease. In: Koopman W. J., ed., ARTHRITIS AND ALLIED CONDITIONS: A TEXTBOOK OF RHEUMATOLOGY, Baltimore: Williams & Wilkins, 13 ed., pp. 255-308 (1997)), thereby limiting the capacity of cartilage repair (Campbell, C. J., The healing of cartilage defects, Clin. Orthop. 64:45-63 (1969); Kim, H. K., et al., The potential for regeneration of articular cartilage in defects created by chondral shaving and subchondral abrasion: an experimental investigation in rabbits, J. Bone Joint Surgery Am. 73:1304-1315 (1991)) and because type II collagen is the most abundant protein of cartilage matrix, the assessment of type II collagen synthesis and degradation is an attractive approach for the investigation of OA and other cartilage degeneration conditions.
In vitro studies performed on cartilage tissue from patients with OA and controls have provided evidence of altered synthesis (Nelson, F., et al., Evidence of altered synthesis of type II collagen in patients with osteoarthritis, J. Clin. Invest. 102:2115-2125 (1998)). Certain immunoassays have indicated increased degradation of type II collagen in OA (Hollander, A. P., et al., Increased damage of type II collagen in osteoarthritic articular cartilage detected by a new immunoassay, J. Clin. Invest. 93:1722-1732 (1994); Billinghurst, R. C., et al., Enhanced cleavage of type II collagen by collagenase in osteoarthritic articular cartilage, J. Clin. Invest. 99:1534-1545 (1997); Hollander, A. P., et al., Damage to type II collagen in aging and osteoarthritis starts at the articular surface, originates around chondrocytes and extends into the cartilage with progressive degeneration, J. Clin. Invest. 96:2859-2869 (1995)). However, molecular markers that indicate both collagen synthesis and degradation have remained unavailable. Therefore, accurate and precise assessment of the level and/or progression of OA and other cartilage degenerative conditions in vitro and/or in vivo has remained a significant problem.
Type II collagen is synthesized as a procollagen molecule including the N-(PIINP) and C-(PIICP) propeptides at each end. Type II procollagen is produced in two forms as the result of alternative RNA splicing (Ryan, M. S., et al., Differential expression of a cystein-rich domain in the amino-terminal propeptide of type II (cartilage) procollagen by alternative splicing of messenger RNA, J. Biol. Chem. 265:10336-10339 (1990); Nah, H. D., et al., Type II collagen mRNA containing an alternatively spliced exon predominates in the chick limb prior to chondrogenesis, J. Biol. Chem. 266:23446-23452 (1991)). One form (IIA) includes and the other form (IIB) excludes a 69 amino acid cysteine-rich globular domain encoded by exon 2 in the PIINP. Type IIB procollagen is expressed at high levels in well-differentiated chondrocytes, forming the framework of normal adult cartilage. On the other hand, type IIA procollagen is temporally expressed in prechondrogenic condensing limb mesenchyme, sclerotome and early cartilage (Sandell, L. J., et al., Alternatively spliced type II procollagen mRNAs define distinct populations of cells during vertebral development: differential expression of the amino-propeptide, J. Cell. Biol. 114:1307-1319 (1991); Sandell, L. J., et al., Alternative splice form of type II procollagen mRNA (IIA) is predominant in skeletal precursors and non-cartilaginous tissues during early mouse development, Dev. Dyn. 199:129-140 (1994); Lui, V. C., et al., Tissue-specific and differential expression of alternatively spliced alpha 1 (II) collagen mRNAs in early embryos, Dev. Dyn. 203:198-211 (1995); Oganesian, A., et al., Type IIA procollagen amino propeptide is localized in human embryonic tissues, J. Histochem. Cytochem. 45:1469-1480 (1997)) and can be re-expressed later in the development at the onset of cartilage hyperthrophy (Nah, H. D., et al., Type IIA procollagen: Expression in developing chicken limb cartilage and human osteoarthritic articular cartilage, Dev. Dyn. 220:307-322 (2001)). In addition it has recently been shown that type IIA procollagen is re-expressed by adult articular chondrocytes of affected human osteoarthritic cartilage (Nah, Dev. Dyn. 220, supra; Aigner, T., et al., Re-expression of type IIA procollagen by adult articular chondrocytes in osteoarthritic cartilage, Arthritis Rheum. 42:1443-1450 (1999)). During secretion and before incorporation of type II collagen molecules into cartilage matrix, the N and C propeptides are removed by specific enzymes and released in part into the synovial fluid and cleared into the blood. The serum level of these propeptides is thus believed to represent an adequate index of the rate of type II collagen synthesis. The first assays developed to investigate type II collagen synthesis were for PIICP. Nelson, et al. (J. Clin. Invest. 102, supra) showed that PIICP is a valid index of the rate of type II collagen synthesis in healthy and OA cartilage and that serum PIICP levels were decreased in patients with OA. An ELISA was developed for measuring specifically the N-propeptide of type IIA procollagen (PIIANP) with no significant cross-reactivity with type I collagen N-propeptide and reported decreased serum levels of PIIANP in patients with knee OA and RA compared to age-matched healthy controls suggesting a deficit of type II collagen synthesis in joint diseases (Rousseau, J-C., et al., Abstract, Serum levels of type II A procollagen amino terminal propeptide (PIIANP) are decreased in patients with knee osteoarthritis and rheumatoid arthritis, Arthritis Rheum. 43 (supp.):S351 (2000)).
To assess type II collagen degradation, immunoassays using antibodies recognizing either neo-epitopes generated by denaturation of the triple helix domain of type II collagen (Hollander, J. Clin. Invest. 93, supra; Downs, J. T., et al., Analyis of collagenase-cleavage of type II collagen using a neoepitope ELISA, J. Immunol. Methods 247:25-34 (2001)) or cross-linked fragments of the telopeptides (Moskowitz, R. W., et al., Abstract, Type II C-telopeptide 2B4 epitope is a marker for cartilage degradation in familial osteoarthitis, Arthritis Rheum. 41 (supp.):S352 (1998); Christgau, S., et al., Collagen type II C-telopeptide fragments as an index of cartilage degradation, Bone 29:209-215 (2001)) have been recently developed. Using an assay recognizing C-terminal cross-linking telopeptide of type II collagen (CTX-II) in urine (Christgau, supra), subjects of a cross sectional study showed increased levels of urinary CTX-II in patients with knee OA (Garnero, P., et al., Cross sectional evaluation of biochemical markers of bone, cartilage, and synovial tissue metabolism in patients with knee osteoarthritis: relations with disease activity and joint damage, Ann. Rheum. Dis. 60:619-626 (2001)).
Some methods of detecting OA are presently known in the art. For example, U.S. Pat. No. 5,780,240 to Sandell, incorporated herein by reference in its entirety, describes assays to detect cartilage synthesis in OA patients. The assays are useful in providing methods for detecting type IIA mRNA and/or type IIA procollagen/propeptide in samples from non-embryonic individuals. However, while the methods allow determination of whether a patient has OA, they do not provide a method for determining the progress of OA in a patient, how to determine the rate of progression of the disease, how to determine the likelihood of increased or decreased OA progress, nor how to determine the efficacy of drugs on the progress of OA. Likewise, U.S. Pat. No. 5,541,066 to Sandell, incorporated herein by reference in its entirety, describes assays for determining cartilage synthesis associated with osteoarthritis, but also fails to overcome the limitations in the art with respect to evaluating the progress of disease in a patient with OA.
A cross-sectional study of multiple (fourteen) molecular markers for monitoring osteoarthritis has been described. The markers of inflammation, bone, cartilage and synovium metabolism were segregated into clusters and used in distinguishing osteoarthritis at baseline. Otterness et al., An analysis of 14 molecular markers for monitoring osteoarthritis: segregation of the markers into clusters and distinguishing osteoarthritis at baseline, Osteoarthritis and Cartilage 8:180-185 (2000). Using a principal component analysis, as opposed to an uncoupling analysis, the study reported that molecular markers reflecting cartilage synthesis (serum aggregan epitope 846 and PIICP) and those of cartilage catabolism (serum cartilage oligomeric matrix protein (COMP) and keratan sulfate) segregated into two separate and independent factors and that the combination of these two groups together with tumor necrosis factor receptor type II (a marker of inflammation) provided the best discrimination between patient with knee OA and healthy controls. However, only three markers (tumor necrosis factor receptor II, cartilage oligomeric matrix protein and epitope 846) from independent clusters minimally discriminated osteoarthritis patients from controls. A conclusion of the study was that better markers are needed to accurately and precisely determine the status of osteoarthritis in subjects.
The use of molecular markers of bone formation and bone resorption to determine type I osteoporosis has been described. Eastell, R. et al, Evaluation of bone turnover in type I osteoporosis using biochemical markers specific for both bone formation and bone resorption, Osteoporos Int. 3:255-260 (1993). However, bone synthesis and degradation markers are not associated with collagen synthesis and degradation markers and are not predictive whether such collagen markers could be used in a predictive way with osteoarthritis, rheumatoid arthritis and other cartilage degeneration conditions.
Accordingly, there is a need for cartilage markers that can provide information on collagen metabolism and the progression of the arthritic disease state. Such markers would be useful in estimating the progression of cartilage degeneration diseases such as OA and RA. In addition, such markers would allow accurate determination of the therapeutic effects certain cartilage degeneration drug treatments, including osteoarthritis and rheumatoid arthritis drug treatments, so would be useful for pharmaceutical efficacy studies in mammals.